The mammary gland is a complex organ whose growth and development are controlled by the interaction of a wide variety of hormones and growth factors also involved in the etiology and progression of the cancerous state. Our emphasis has been on the interactions of prolactin (PRL), estrogen (E), and progesterone (P) during the peripubertal period and the lobulo-alveolar development of pregnancy as well as during tumorigenesis. We have shown that E and P are required to promote development of the primary/secondary ductal network in addition to other endocrine growth factor(s), and that P facilitates the formation of tertiary side-branches. The Hox-related homeobox containing gene, Msx2, is highly expressed during branching morphogenesis where our studies in vivo and in vitro showed that its expression is regulated by P in the presence of E. Concurrent with these morphological changes, progesterone receptor (PR) localizes at early branch points. During peripubertal morphogenesis PR distribution shifted from a homogeneous to a heterogeneous pattern. Concomitantly the PRL receptor (PRLR) undergoes a similar shift in pattern. The transcription factor, C/EBP-beta appears to regulate mammary epithelial cell fate resulting in the correct spatial pattern of gene expression required to permit steroid hormone regulated cell proliferation.We demonstrated differential transcription of the four PRLR isoforms by stromal as well as epithelial cells throughout development. The distribution of the PRLR in the epithelium, like that of the PR, progressed from a homogeneous to a heterogeneous pattern. Hence, while exogenous P or PRL alone was without effect on epithelial proliferation in ovariectomized mice, these hormones synergize to stimulate epithelial and stromal proliferation. We have also identified that insulin-like growth factor-II (IGF-II) may mediate PRL-induced proliferation in the mammary gland. Expression of IGF-II is increased in ductal and alveolar epithelium during puberty- and pregnancy-associated morphogenesis, and IGF-II stimulates lobuloalveolar development in whole organ culture. We are studying changes in the vascular network that facilitates lactogenesis and tumorigenesis in the mammary gland. Our data support the conclusion that specific cell types within the mammary gland differentially transcribe VEGF and that it functions as an autocrine/paracrine endothelial growth factor under hormonal regulation. Additional studies aim to understand the role of PRL in the etiology and progression of human breast cancer. Specifically, we are examining the role of PRLR isoforms and autocrine/paracrine PRL in tumorigenesis and carcinogenic susceptibility. Comparisons between cancerous and adjacent, noninvolved tissue from the same breast of 23 patients showed that, on average, both PRL and PRLR mRNA expression was significantly higher in the cancerous tissue compared to the noninvolved tissue. The various forms of the PRLR differ in their cytoplasmic domains due to alternate splicing. Using 3' RACE we isolated five splice variants of the hPRLR, three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9, respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.